Sirtuin6對類風濕性關節炎進展的可能調控機制

Abstract

類風濕性關節炎(Rheumatoid arthritis)為一自體免疫所引起的全身性發炎反應，主要發生於滑液關節腔處因持續之慢性發炎導致硬骨及軟骨吸收破壞。此疾病之盛行率約為1%，台灣約有十萬人罹患此病。 Cyr61(Cystine-rich angiogenic inducer 61)又稱為CCN1，為CCN family成員之一，為一分泌性蛋白，已知與血管新生，發炎反應，細胞及腫瘤生長相關。文獻中發現Cyr61於類風濕性關節炎患者的B細胞中會有不正常的大量表現，Cry61同時也可吸引發炎細胞與巨噬細胞聚集以及促進血管新生，然而確切的作用機制目前仍不清楚，因此我們希望能進一步了解Cyr61在關節炎中扮演的角色。 我們的前導實驗中顯示，Cyr61可促進Monocyte的細胞驅性，當成骨細胞株(U2OS cell, a osteosarcoma cell line with osteoblastic phenotype)接受Cyr61刺激後，Monocyte chemoattractant protein 1(MCP-1)的表現會增加。Cyr61我們的前導實驗亦證實於U2OS細胞中CREB()可以調控Cyr61基因的表現。 SIRT6為Sirtuin family之七種異位體(isoform)之一，Sirtuin family為一群NAD+ dependent / nondependent protein deacetylase，大多於acetyl-lysine處進行去乙醯化的作用，已知作用於組蛋白(Histone)上時可調節不同的基因轉錄作用。SIRT family已知與抗老化，抗發炎相關，SIRT6與發炎反應的相關研究並不多，2009年有文獻提及SIRT6可藉由抑制Histone H3處之乙醯化影響NFkB pathway –一個已知與Aging, Inflammation相關的signaling pathway。因此我們對於SIRT6是否可以調控Cyr61的表現感到高度的興趣。 本實驗採取臨床上判別為Rheumatoid arthritis的病理組織切片做免疫組織染色，觀察Cyr61與SIRT6表現的相關性。In vitro則以U2OS(osteosarcoma cell line with osteoblast phenotype)作為實驗材料，並建立一個持續表現SIRT6的 U2OS cell line，觀察於TNFα刺激下，SIRT6的存在是否可調控Cyr61與CREB的表現，並以Luciferase assay觀察CREB與Cyr61 promoter上之CRE結合能力是否受SIRT6影響。In vivo的部份則觀察在以建立的Rat CIA(collagen induced arthritis) model中Cyr61與SIRT6表現的關連性。 實驗結果顯示表現SIRT6的stable cell受到刺激後Cyr61的表現量較低，接受刺激後CREB磷酸化的趨勢亦較不明顯，SIRT6亦會降低Cyr61 promoter activity，而這樣的趨勢在以site-directed mutagenesis將Cyr61 promoter上之CRE置換後變的不明顯，顯示CREB可能為SIRT6調控Cyr61的一中間途徑；而在組織中可以見到Cyr61於發炎嚴重的區域有較強烈的表現，而SIRT6多表現於進行regeneration的組織之中，兩者於組織中的表現呈現一相反的趨勢。 結論: In vivo中，SIRT6會透過CREB(cAMP responsive element binding protein)來調控Cyr61的表現。In vitro中則可見到Cyr61於發炎強烈的組織中表現較明顯，而SIRT6則多表現於較活耀之新生骨細胞中，兩者之間之相互關聯則待更進一步之實驗證實。

Rhumatoid arthritis (RA) is a systemic, chronic autoimmune disorder, and is characterized by progressive destruction of normal joint soft tissue, bone erosion, and eventually joint deformity. The prevalence of RA in temporalmandibular joint is about 14~85%, and there are about 100,000 affected cases in Taiwan. Cyr61, a cystine-rich protein, found to be play an important role in RA patients. CCN1 was known to be associated with angiogenesis, inflammation, cell and tumor growth. Literature review showed compared with the healthy co-twin, higher level of Cyr61 in RA patients with cDNA micro-array analysis of B cells. Cyr 61 also recruit inflammatory cell & marchpharge to inflammatory sites, induce angiogenesis, but the mechanism still remained unknown. In literature review, Cyr61 may stimulates Monocyte chemoattractant protein 1 (CCL2/MCP-1) to promote macrophage hemotaxis was noticed. Our previous experients also showed that Cyr61 can enhance CCL2/MCP-1 expression in U2OS cells (a a osteosarcoma cell line with osteoblastic phenotype). Our previous data also showed that Cyr61 expression may be regulated via phosphoraton of CREB(c-AMP responsive element binding protein) SIRT6, a member of the sirtuin (SIRT) family, is a NAD-dependent deacetylase that promote longevity in multiple organisms. SIRT may regulate different gene expression through interaction with histone. Lack of SIRT6 leads to shortened life span and an aging-like phenotype in mice. Kawahara et al. noticed that SIRT6 attenuates NF-kB signaling through histone deacetylation (cell 2009). NFκB, a signaling pathway known to be associated with aging and inflammation, indicated that SIRT6 is thought to be related with inflammation and we wondered if SIRT6 could regulate Cyr61 expression. Human specimen of RA patient were examined with Immunochemistry stain. To examine the effects of pro-inflammatory cytokines on Cyr61 expression in osteoblastic cells and the modulatory action of SIRT6, A U2OS cell stable clone regularly expressed SIRT6 was selected. The role of cAMP response element (CRE)-binding protein (CREB) in Cyr61 induction was assessed, and western blot were used to check the expression level of Cyr61 and Phospho-CREB. Luciferase assay was used to check the Cyr61 activity; ELISA for secreted MCP-1 were also examinated. In a rat model of collagen-induced arthritis (CIA), the relation of osteoblastic expression of Cyr61 and SIRT6 to disease progression was evaluated. RESULT: Lower Cyr61 expression level were noticed after treated with TNFa in SIRT6-expressing U2OS group, and lower phosphor-CREB level were also noticed. In luciferase assay, SIRT6 also lower Cyr61 promoter activity, indicates that SIRT6 regulates Cyr61 expression through CREB phosphorylation. In Human RA tissue andrat CIA model, Cyr61 were found in tissue with more severe inflammation activity, and SIRT6 were found in tissue with a bone-remodeling pattern. There seemed to be a opposite tendency between SIRT6 and Cyr61 expression. CONCLUSION: In vitro, SIRT6 regulates Cyr61 expression via inhibit CREB activation; in vivo, Cyr61 were expressed in tissue with more severe inflammation and SIRT6 were found in tissue with a higher bone remodeling ability. The interaction between Cyr61 and SIRT6 needs further investigation.